活性氧ROS对小鼠早期胚胎发育阻滞的影响研究

发布时间：2018-05-01 05:36

本文选题：线粒体膜电位 + mtDNA拷贝数　；参考：《内蒙古大学》2017年硕士论文

【摘要】：哺乳动物胚胎体外培养体系影响着早期胚胎的发育潜能。在目前模拟体内输卵管及子宫的低氧环境的培养系统中,活性氧(reactive oxygen species,ROS)被认为是影响胚胎发育的一个重要因素。胚胎合子基因组激活(zygotic gene activation,ZGA)是早期胚胎正常发育的关键,研究认为ZGA的延迟或失败与早期胚胎发育阻滞有关。为探究ROS对小鼠早期胚胎发育阻滞的影响,本研究比较了不同品种小鼠在不同培养液中发生阻滞的情况,检测了 ZGA相关基因、胞质ROS浓度及ROS相关基因的表达;同时利用2,2-偶氮二(2-甲基丙基咪)二盐酸盐AAPH处理昆明白小鼠体外受精卵建立ROS引起阻滞的细胞模型,对线粒体机能相关的线粒体膜电位、线粒体ROS(mtROS)及线粒体分布状态、mtDNA的拷贝数进行了检测;另外,检测了线粒体相关基因Grm2、Drd2、Polg2、TE4M的表达变化及其调节区的DNA甲基化的调节。1、不同品系小鼠胚胎在不同培养液中合子基因组激活与ROS相关性研究本研究利用实时荧光定量PCR的方法检测了小鼠2-细胞胚胎中ZGA相关基因(Zscan4、MuERV-L、Eif-1a、Hsp70.1)及ROS相关基因(Nox1、Gpx4、Gpu6、Prdx2)的表达,并利用ROS特异性染料DCFH-DA对胚胎胞质内ROS水平进行检测。首先比较了阻滞品系小鼠KM与非阻滞品系小鼠BDF1受精卵在M16培养液中发生阻滞的情况,结果表明,在M16培养液中,KM小鼠受精卵的4-细胞发育率为44.2%,而BDF1小鼠受精卵的发育率为90.3%,KM小鼠2-细胞胚中ZGA相关基因MuERV-L、Eif-1a的表达量显著低于BDF1小鼠2-细胞胚(P0.05)。KM小鼠2-细胞胚胎的胞质内ROS水平明显低于BDF1胚胎,ROS相关基因Gp和Prdx2的表达显著高于BDF1小鼠。以上研究表明,小鼠胚胎发育阻滞与ZGA相关基因表达有关,ROS水平的差异反映出不同品系小鼠胚胎对ROS的耐受性不同。之后利用M16培养液和KSOM培养液比较了阻滞品系KM小鼠胚胎在不同培养液条件下的发育。结果显示,在KSOM培养液中,KM小鼠受精卵的4-细胞率为90.8%,不发生2-细胞阻滞。KSOM培养液中培养的KM小鼠2-细胞胚胎Eif-1a、Hsp70.1表达量显著高于M16培养液中培养的2-细胞胚胎。两种培养液中的KM小鼠2-细胞胚胎的胞质内ROS水平和ROS相关基因表达无明显差异。以上研究表明,小鼠胚胎体外培养液成分的不同影响ZGA相关基因表达的变化,对小鼠胚胎发育阻滞产生影响。2、ROS对发育阻滞胚胎中线粒体相关机能的影响本实验探究了 ROS在小鼠体外发育2细胞阻滞胚胎中对线粒体相关机能的影响。使用AAPH处理昆明白小鼠体外受精卵,通过荧光探针检测处理组和对照组中线粒体膜电位、线粒体ROS(mtROS)及线粒体分布状态的变化,并利用实时荧光定量PCR检测ROS相关基因、线粒体相关基因(Grm2、Drd2、Polg2、TFAM)mRNA的表达量及mtDNA的拷贝数,利用重亚硫酸盐测序(BSP)检测了线粒体四个相关基因的甲基化变化。研究结果显示,使用1.0 mmol/L浓度的AAPH分别处理的胚胎,具有较高的阻滞率(66.16%)和较低的损伤率(24 h畸形率9.09%,48 h死亡率10.39%),ROS染色实验结果显示,AAPH处理能够使胚胎内胞质ROS水平明显升高,所检测的ROS相关基因mRNA表达量升高。1.0 mmol/L处理后二细胞期胚胎线粒体膜电位及mtROS水平明显高于对照组,mtDNA的拷贝数增加,实时荧光定量PCR结果显示,处理组与对照组的Grm2、Drd2、Polg2、TFAM基因表达量呈现显著性差异,处理组Grm2、Drd2表达显著低于对照组,处理组Polg2、TFAM表达显著高于对照组。利用亚硫酸盐测序(bisulfite genomic sequencing,BSP)方法检测DNA甲基化的结果,AAPH处理之后的胚胎中基因调节区DNA甲基化水平升高;Polg2、TFAM甲基化水平降低。以上研究表明,AAPH处理引起的氧化应激反应及发育阻滞的胚胎中,线粒体活性升高,线粒体数目增加,线粒体相关基因表达变化,表明线粒体参与调节细胞内ROS的动态平衡,ROS可能通过影响线粒体相关基因启动子区DNA甲基化的变化调控线粒体相关基因的时序性表达。综上所述,不同品系小鼠胚胎对ROS的耐受性不同,体外培养环境中小鼠胚胎发育阻滞与ZGA相关基因表达有关,线粒体参与调节细胞内ROS的动态平衡影响胚胎发育。本研究初步探究了 ROS与线粒体相关功能变化的关系,为优化哺乳动物胚胎体外培养体系提供参考。
[Abstract]:In vitro culture system of mammalian embryos affects the developmental potential of early embryos. Reactive oxygen species (ROS) is considered as an important factor affecting embryo development in the culture system that simulates the hypoxic environment of the oviduct and uterus in the body at present. The genome activation of the embryo of the embryo (zygotic gene activation, ZGA) is early. The key to the normal development of the embryo is that the delay or failure of ZGA is related to the early embryonic development block. In order to explore the effect of ROS on the early embryonic development block of mice, this study compared the arrest of different mice in different cultures, and detected the ZGA phase gene, the concentration of cytoplasmic ROS and the table of ROS related genes. At the same time, using 2,2- azo two (2- methyl proprom) two hydrochloride AAPH to treat the cell model of ROS block in vitro fertilized eggs of Kunming white mice, the mitochondrial membrane potential related to mitochondrial function, mitochondrial ROS (mtROS) and mitochondrial distribution state, and the number of mtDNA copy scallop were detected, and the mitochondrial related gene Gr was detected. The expression changes of M2, Drd2, Polg2, TE4M and the regulatory.1 of DNA methylation in the regulatory region, the study on the correlation between the activation of zygote genomes in different strain mice embryos and the correlation of ROS in different cultures. This study used real time fluorescence quantitative PCR to detect ZGA phase Guan Jiyin in mouse 2- cell embryos. The expression of the gene (Nox1, Gpx4, Gpu6, Prdx2) and the ROS specific dye DCFH-DA were used to detect the ROS level in the cytoplasm of the embryo. First, the arrest of the BDF1 fertilized eggs of the block mouse KM and the non block strain mice in the M16 culture solution was compared. The results showed that the growth rate of the fertilized egg of the KM mice was 44. in the M16 culture medium. 2%, the growth rate of the fertilized eggs of BDF1 mice was 90.3%, and the ZGA related gene MuERV-L in the 2- cell embryos of the KM mice was significantly lower than that of the BDF1 mouse 2- cell embryos (P0.05).KM mice. The ROS level in the cytoplasm of the 2- cell embryos was significantly lower than that of the embryos. The embryonic development block was related to the expression of ZGA related genes. The difference of ROS level reflected the different tolerance of mouse embryos from different strain mice to ROS. Then the development of KM mouse embryos in the block line was compared with the M16 culture and KSOM culture. The results showed that the 4- cells of the fertilized eggs of KM mice were in the KSOM culture solution. The rate was 90.8%, the KM mouse 2- cell embryo Eif-1a was not cultured in the.KSOM culture medium with 2- cell block. The expression of Hsp70.1 was significantly higher than that of the 2- cell embryos cultured in the M16 culture solution. The ROS level and the ROS related gene expression in the cytoplasm of the KM mouse 2- cell embryos in the two cultures were not distinct. The above study showed that the mouse embryos were in vitro The effects of the different components of the culture medium on the expression of ZGA related genes, the effect of.2, and the effect of ROS on the mitochondrial related function in the developmental block embryos, the effect of ROS on the mitochondrial related function in the 2 cell block embryos developed in vitro in mice was investigated. The use of AAPH in the treatment of Kunming white mice in vitro was studied. Sperm egg, the mitochondrial membrane potential, mitochondrial ROS (mtROS) and mitochondrial distribution in the treatment group and the control group were detected by the fluorescence probe, and the ROS related genes were detected by real-time fluorescence quantitative PCR, the amount of mRNA and the copy number of mtDNA of the mitochondrial related genes (Grm2, Drd2, Polg2, TFAM) were detected by the heavy sulphite sequencing (BSP) detection. The change of methylation of four related genes of mitochondria was observed. The results showed that the embryos treated with 1 mmol/L concentration of AAPH had higher block rate (66.16%) and lower damage rate (24 h malformation rate 9.09%, 48 h mortality 10.39%). The results of ROS staining showed that AAPH treatment could significantly increase the ROS level in the cytoplasm of the embryo. The expression of ROS related gene mRNA increased by.1.0 mmol/L. The mitochondrial membrane potential and mtROS level of the two cell stage embryos were significantly higher than those of the control group. The copy number of mtDNA increased. The real-time fluorescence quantitative PCR results showed that the Grm2, Drd2, Polg2, and TFAM base of the treated group and the control group showed significant difference. The expression of Polg2 and TFAM in the treatment group was significantly higher than that in the control group. The results of DNA methylation were detected by the method of bisulfite genomic sequencing (BSP), and the level of DNA methylation in the gene regulatory region of the embryos after AAPH treatment was increased; Polg2, TFAM methylation level decreased. The above study showed that AAPH treatment caused it. In the oxidative stress reaction and the development block embryo, the mitochondrial activity is elevated, the number of mitochondria is increased, and the mitochondrial related gene expression changes, indicating that mitochondria are involved in regulating the dynamic balance of ROS in cell, and ROS may regulate the sequence of mitochondrial related genes by affecting the changes of DNA methylation in the promoter region of the mitochondrial related genes. To sum up, the tolerance of mouse embryos in different strains of ROS was different. The development of mouse embryo retardation was related to the expression of ZGA related genes in the culture environment. Mitochondria involved in regulating the dynamic balance of ROS in the cell. This study explored the relationship between ROS and mitochondrial related function, in order to optimize the mammal. It provides reference for the culture system of embryo in vitro.

【学位授予单位】：内蒙古大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q954.4

【参考文献】